Ubiquitin (Ub) is a highly conserved 8.5 kD protein that is unusual because it is found both free and covalently linked to other proteins cells. It is involved in fundamental cellular events and with the aging process. Defects in Ub functions have been implicated in a variety of disorders including muscular dystrophy and Alzheimer's disease. Although its functions are unknown, Ub appears to be involved in the degradation of defective proteins. It is also conjugated to a set of specific and relatively stable proteins (histones and growth factor receptors). We discovered that Ub has intrinsic proteolytic activity and that it may exist in several different structural states. These findings provide a rational approach to discerning the functions and the mechanisms by which it participates in cellular events. To that end, we have developed a set of immunochemical reagents to Ub, protein microanalytical techniques, and an in vitro assay system for the intrinsic proteolytic activity. Specifically, we will: 1) define the factors affecting the proteolytic activity of free Ub. Ub expressed on a vector in E. coli will be purified and the effect of site specific mutations on proteolytic activity will be examined. The cleavage specificity of the Ub protease will be determined and used to design specific substrates and inhibitors. Regulators of its proteolytic activity will be identified 2) test the hypothesis that Ub is active as a multimer and examine the structural organization in vitro. Sizing techniques, specific antibody probes, chemical x-linking and surface modification will be used to study the structural organization of Ub by comparing active, inactive and genetically altered forms; and 3) test the hypothesis that Ubiquitination of a specific protein creates a new protease (the ad hoc protease model). The proteolytic activity of Ub in specific conjugates will be characterized. The attachment site in naturally occurring conjugates (e.g. PDGF receptor) will be determined so that site-specific mutagenesis can be used to critically examine its physiologic functions in vivo. Our studies will provide insight into Ub's functions and its roles in cellular events.